Sulphated vinyl polymers in compositions for treating retroviral infections

ABSTRACT

Sulphated vinyl polymers such as sulphated polyvinylalcohol, sulphated copolymers of (meth)acrylic acid and vinylalcohol, and pharmaceutically acceptable salts thereof, have an antiviral effect against human immunodeficiency virus and may therefore be used as a therapeutic agent for treating retrovirus infections like AIDS and AIDS-related diseases. Their antiviral activity is apparently based on inhibition of virus adsorption; their inhibitory effect on giant cell formation is stronger than that of dextran sulphate.

This invention relates to a novel therapeutic agent for treatingretroviral infections such as AIDS and AIDS-related diseases.

AIDS or acquired immune deficiency syndrome is a pandemicimmunosuppressive disease resulting from the depletion of helperT-lymphocyte cells in the human body. The causative agent has beenidentified as a retrovirus called "human immunodeficiency virus" or HIV.At the moment, two different types (HIV-1 and HIV-2) of that retrovirushave been described; both types can induce AIDS or AIDS-related diseasesalthough HIV-1 is more widely spread than HIV-2.

Many attempts have already been made to find suitable anti-HIV agentsand various substances and compounds have been reported to inhibit thereplication of HIV (usually type 1) in vitro.

Among the suggested anti-HIV compounds, 3'-azido-2',3'-dideoxythymidine(azidothymidine or AZT) is at the moment the only compound that hasproved to be clinically effective in the treatment of AIDS patients,compare Fischl et al, New Engl. J. Med. 317, 185-191 (1987). Theactivity of AZT (after conversion to its 5'-triphosphate form) isapparently based on inhibition of reverse transcriptase, an enzymeplaying a part in the replication of HIV-1 in T-lymphocyte cells.

Further, sulphated polysaccharides such as dextran sulphate, heparin andpentosan polysulphate, are highly potent and selective inhibitors ofHIV-1 replication during tests in vitro. Compare:

Ito et al, Antiviral Res. 7, 361-367 (1987),

Baba et al, Proc. Nat. Acad. Sci. USA, 85, 6132-6136 (1988) and

Baba et al, Antiviral Res. 9, 335-343 (1988).

The activity of these sulphated polysaccharides is apparently based oninhibition of the adsorption of virus particles onto the cell membranesof T-lymphocyte cells.

In addition to this known matter, there is need for other substances andcompounds acting as potent HIV-inhibitors. Therefore, the invention hasfor its primary object to provide such inhibitors as well as therapeuticcompositions containing the same.

In accordance with the invention, it has now been found that sulphatedvinyl polymers such as sulphated polyvinylalcohol and sulphatedcopolymers of (meth)acrylic acid with vinylalcohol as well aspharmaceutically acceptable salts thereof, are highly potent andselective inhibitors of both HIV-1 and HIV-2 replication in vitro andthat they may therefore be used with advantage in pharmaceuticalcompositions intended for the treatment of retroviral infections such asAIDS and AIDS-related diseases.

The term "sulphated polyvinylalcohol" as used herein refers to a groupof polymers of varying molecular weight and polymerisation degree, whichhave a good solubility in aqueous fluids and which are chemicallycharacterized by the presence of a plurality of --OSO₃ H groups togetherwith some --OH groups pending from a normal hydrocarbon (polyvinyl)backbone. They can be synthesized by polymerising vinyl acetate or anyother hydroxyl-protected vinylalcohol derivative followed bydeprotection of the hydroxyl groups in the resulting polymer and bysulphation of the free hydroxyl groups. The sulphation degree can haveany value between 50% and 100%.

The term "sulphated copolymers of (meth)acrylic acid with vinylalcohol"as used herein refers to a group of polymers of varying molecular weightand polymerisation degree, which have a fair solubility in aqueousfluids and which are chemically characterized by the presence of aplurality of --OSO₃ H groups together with some free --OH groups as wellas --COOH groups and optionally methyl groups pending from a normal(polyvinyl) hydrocarbon backbone. They can be synthesized bycopolymerising acrylic or methacrylic acid or reactive derivativesthereof with a hydroxyl-protected vinylalcohol derivative, followed bydeprotection of the hydroxyl groups and, if necessary, the carboxylgroups in the resulting copolymer and by sulphation of the free hydroxylgroups. Again the sulphation degree may have any value between 50% and100%.

The --OSO₃ H groups and --COOH groups (if present) may sometimes existin the form of pharmaceutically acceptable salts; such salts can also beused in the present invention.

The term "sulphated vinyl polymers" is used in this specification todenote the whole group consisting of sulphated of polyvinylalcohols,sulphated copolymers of (meth)acrylic acid and vinylalcohol andpharmaceutically acceptable salts thereof.

It should be noted that such sulphated vinyl polymers differ fromsulphated polysaccharides by the fact that they are substantially freeof cyclic groups in the hydrocarbon backbones of their molecules.

The sulphated vinyl polymers are known compounds. Sulphatedpolyvinylalcohols have been disclosed by Chargaff et al in J. Biol.Chem. 115, 160 (1936) and are commercially available. Sulphatedcopolymers of (meth)acrylic acid and vinylalcohol and their salts havebeen disclosed in EP-A-0093489 where they are intended for use asanticoagulants.

In tests leading to the invention, it was found that the sulphated vinylpolymers completely inhibited the cytopathogenicity of HIV-1 in MT-4cells and the antigen expression of HIV-1 in CEM cells, at aconcentration of 0.8 μg/ml. They were about equally active against HIV-2replication. This is an important fact because the cytopathogenicity ofHIV resulting from its replication in T-cells is normally regarded asthe major mechanism for destruction of such cells.

Moreover, and in contrast with azidothymidine, the sulphated vinylpolymers completely inhibited the HIV-1 induced formation of giant cells(multinucleated syncytium cells) from uninfected T-cells, at aconcentration of 4 μg/ml. This is important because giant cell formationresulting from the interaction between the gp120 surface antigens of theHIV-infected cells and the CD4 receptor of the uninfected cells isanother possible mechanism for the destruction of the T-cells.

Thanks to the inhibition of giant cell formation, the sulphatedvinylpolymers of the present invention have a clear advantage overazidothymidine which does not show such effect.

Further, the sulphated vinyl polymers of the invention were found tocompletely inhibit the adsorption of HIV-particles to CD4 positivecells, at a concentration of about 4 μg/ml. This may account for thestrong activity of these polymers against HIV replication.

When the sulphated vinyl polymers of the invention are compared withsulphated polysaccharides such as dextran sulphate, it appears that bothgroups of compounds have the capability of inhibiting HIV replication,inhibiting giant cell formation and inhibiting virus adsorption toT-cells. Nevertheless, there is a clear difference in activity betweenthese two groups of compounds.

In comparative tests on the inhibition of giant cell formation, it wasfound that dextran sulphate could completely suppress the giant cellformation only at a concentration of 100 μg/ml. Such a concentration isextremely high and hardly achievable in human plasma.

On the other hand, the sulphated vinyl polymers of the present inventionwere capable of completely blocking the giant cell formation at aconcentration of 4 μg/ml, which concentration can be readily achieved.

Thus, the sulphated vinyl polymers have a clear advantage over sulphatedpolysaccharides like dextran sulphate.

Therapeutic compositions, containing the sulphated vinyl polymers of theinvention as an active ingredient for the treatment of retroviralinfections such as AIDS or AIDS-related diseases in human practice maytake the form of powders, suspensions, solutions, sprays, emulsions,unguents or creams and may be used for local application, forintranasal, rectal, vaginal and also for oral or parenteral(intravenous, intradermal, intramuscular, intrathecal etc.)administration. Such compositions may be prepared by combining (e.g.mixing, dissolving etc.) the sulphated vinyl polymers withpharmaceutically acceptable excipients of neutral character (such asaqueous or non-aqueous solvents, stabilizers, emulsifiers, detergents,additives) and further, if necessary with dyes and aromatizers. Theconcentration of the active ingredient in the therapeutic compositionmay vary widely between 0.1% and 100%, depending on the mode ofadministration. Further, the dose of the active ingredient to beadministered may vary between 0.1 mg and 100 mg/kg of body weight.

The anti-HIV properties of the sulphated vinyl polymers are documentedby the following examples which should not be read in a restrictingsense. Dextran sulphate is used for comparison therein.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a diagrammatic representation of the results of tests on theinhibition of cytopathogenicity of HIV-1 together with test results onthe inhibition of virus antigen expression.

FIG. 2 is a graphical representation of the results of tests on theinhibition of giant cell formation.

FIG. 3 is a graphical representation of the results of tests on theinhibition of HIV-adsorption.

In the Examples, reference is made to FIGS. 1-3 of the attached drawing.The viruses used in the examples were HIV-1 and HIV-2. They wereobtained from the culture supernatant of HUT-78 cells persistentlyinfected with HIV-1 and from the culture supernatant of CEM cellspersistently infected with HIV-2, respectively. Titers of HIV-1 andHIV-2 stocks were 2×10⁵ and 5×10⁴ CCID₅₀ (one CCID₅₀ being the 50% cellculture infective dose) per ml, respectively.

The cells used in the Examples were MT-4, CEM and Molt-4 (clone No. 8)cells. The MT-4 cells, disclosed by I. Miyoshi et al., Gann Monogr. 28,219-228 (1982) were used for tests on the inhibition of thecytopathogenicity of HIV and tests on the inhibition of virusadsorption. The CEM cells, disclosed by Foley et al., Cancer, 18,522-529, (1965), were used for tests about the inhibition of viralantigen expression, whereas Molt-4 (clone No. 8) cells, disclosed byKikukawa et al., J. Virol. 57, 1159-1162 (1986) were used in the giantcell formation assay. All cells were mycoplasmanegative. The cells weregrown and maintained in a culture medium consisting of RPMI 1640 mediumsupplemented with 10% heat-inactivated fetal calf serum, 100 U/mlpenicillin G and 20 μg/ml gentamicin. This RPMI 1640 medium is astandard medium comprising inorganic salts like NaCl, NaHCO₃, Na₂ HPO₄etc. as well as glucose, several amino acids and several vitamins.

Four different substances were used as test compounds, namely:

PAVAS (MW 10,000), a sulphated copolymer of acrylic acid andvinylalcohol (monomer ratio 1:9) having a molecular weight of 10,000 anda sulphation degree of 60%;

PAVAS (MW 20,000), a sulphated copolymer of acrylic acid andvinylalcohol (monomer ratio 1:9) having a molecular weight of 20,000 anda sulphation degree of 50%;

PVAS (MW 20,000), a sulphated polyvinylalcohol of molecular weight20,000 and a sulphation degree of 50%;

Dextran sulphate (MW 5,000), a sulphated dextran having a molecularweight of 5,000 and a sulphation degree of about 67%.

EXAMPLE 1 Inhibition of Cytopathogenicity of HIV-1

The test compounds were evaluated for their inhibitory effect on thecytopathogenicity of HIV-1 in MT-4 cells. The method has been describedby R. Pauwels et al. in J. Virol. Methods, 16, 171-185 (1987).

In a first series of tests, MT-4 cells were suspended in culture mediumat 3×10⁵ cells/ml and infected with HIV-1 at 1,000 CCID₅₀ /ml.Immediately after infection, 100 μl portions of the cell suspension wereplaced into the wells of a microtiter® tray containing variousconcentrations of the test compounds. After 5 days incubation at 37° C.,the number of viable cells in each well was determined by means of acolorimetric assay (MTT method), using3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide as acolorant. This colorimetric assay has been described by Pauwels et al inJ. Virol. Methods, 20, 309-321 (1988).

A second series of tests, parallel to the first, was effected withmock-infected cells, incubated in the presence of various concentrationsof the test compounds. A control test was effected by incubatingmock-infected cells in the absence of any test compound.

The results of these tests are represented by the bar diagrams of FIG.1, obtained by plotting the number of viable cells after 5 daysincubation (in percent of the number in the control test) against theconcentration of the test compound in each test. Black bars refer totests with virus-infected cells and white bars refer to tests withmock-infected cells. Panels A, B and C relate to tests with PAVAS (MW10,000), PAVAS (MW 20,000) and PVAS (MW 20,000) respectively.

It can be deduced from FIG. 1 that all three test compounds completelyprotected the cells against virus-induced cell destruction at aconcentration of 0.8 μg/ml.

None of the three test compounds reduced the viability of mock-infectedMT-4 cells at concentrations up to 2500 μg/ml. Thus, the 50% cytotoxicdose (CD₅₀) of the compounds for MT-4 cells was higher than 2500 μg/ml.

EXAMPLE 2 Inhibition of Viral Antigen Expression

The test compounds were also evaluated for their inhibitory effect onthe expression of viral antigens in HIV-infected CEM cells. Here, CEMcells were suspended in the culture medium at 1×10⁵ cells/ml andinfected with HIV-1 at a multiplicity of infection of 0.04. The infectedcells were incubated in the presence of various concentrations of thetest compound. After 4 days incubation, the viral antigen expression wasmeasured by indirect immunofluorescence and laser cytofluorography,using polyclonal antibody as a probe.

The method has been described by Pauwels et al. in J. Virol. Methods,16, 171-185 (1987).

A control test was effected in the absence of any test compound.

The results of these tests have been indicated in FIGS. 1A, 1B and 1C bymeans of curves (- ) obtained by plotting the number of antigen positivecells (in percent of the number of untreated virus-infected cells)against the concentration of the test compound in each test. Again,FIGS. 1A, 1B and 1C relate to tests with PAVAS (MW 10,000), PAVAS (MW20,000) and PVAS (MW 20,000), respectively.

It appears from the curves in FIGS. 1A, 1B and 1C that the three testcompounds almost completely inhibited the viral antigen expression ofHIV-1 in CEM cells at a concentration of 0.8 μg/ml and higher.

During a parallel series of tests, it appeared that none of the testcompounds reduced the viability of mock-infected CEM cells atconcentrations up to 500 ug/ml. Thus, the cytotoxic dose (CD₅₀) of thetest compounds for CEM cells was higher than 500 ug/ml.

EXAMPLE 3 Inhibition of HIV-Induced Cytopathogenicity and of HIV AntigenExpression

The tests of Examples 1 and 2 were expanded to cover both HIV-1 andHIV-2 as test viruses and both MT-4 and CEM cells as test cells.Further, PAVAS (MW 10,000), PAVAS (MW 20,000), PVAS (MW 20,000) as wellas Dextran sulphate (MW 5,000) were used as test compounds.

The results, expressed in values for ED₅₀ (the 50% antivirally effectivedose) and CD₅₀ (the 50% cytotoxic dose) are shown in Table 1 where alldata are mean values of two separate experiments.

                  TABLE 1                                                         ______________________________________                                                       ED.sub.50 (μg/ml)                                           Compound     Cell    HIV-1    HIV-2 CD.sub.50 (μg/ml)                      ______________________________________                                        PAVAS (MW: 10,000)                                                                         MT-4    0.15     0.29  >2500                                                  CEM     0.10     0.14   >500                                     PAVAS (MW: 20,000)                                                                         MT-4    0.11     0.31  >2500                                                  CEM     0.14     0.19   >500                                     PVAS (MW: 20,000)                                                                          MT-4    0.18     0.28  >2500                                                  CEM     0.23     0.39   >500                                     Dextran sulphate                                                                           MT-4    0.39     0.07  >2500                                     (MW: 5,000)  CEM     1.40     0.12   >500                                     ______________________________________                                    

It can be deduced from Table 1 that the antivirally effective doses ofall four test compounds are far below the cytotoxic doses thereof.Further, it can be seen that PAVAS and PVAS are equally inhibitory toHIV-2 and HIV-1 replication in both MT-4 and CEM cells. PAVAS and PVASare slightly more active than dextran sulphate against HIV-1 andslightly less active than dextran sulphate against HIV-2.

EXAMPLE 4 Inhibition of Giant Cell Formation

Molt-4 cells were co-cultured with an equal number (5×10⁴) ofHIV-1-infected HUT-78 cells in the wells of a microtiter® traycontaining various concentrations of the test compounds. After 24 hoursof incubation, the cell clusters were broken by gentle pipetting and thenumber of giant cells (multinucleated syncytial cells) was recordedmicroscopically. A control test was effected in the absence of any testcompound.

The control test revealed the formation of giant cells. Such giant cellswere not observed when the Molt-4 cells had been co-cultured withuninfected HUT-78 cells.

The results of the tests with various concentrations of the testcompounds are graphically represented in FIG. 2 where the number ofgiant cells after 24 hours incubation (in percent of the number in thecontrol test) has been plotted against the concentration of the testcompound in each test.

The curves in FIG. 2 can be identified as follows:

- PAVAS (MW 10,000)

- PAVAS (MW 20,000)

- PVAS (MW 20,000)

◯-◯ Dextran sulphate (MW 5,000)

It appears from FIG. 2 that giant cell formation could be completelyinhibited by PAVAS and PVAS at concentrations of 4 ug/ml. Even at alower concentration of 0.8 μg/ml, PAVAS (MW 10,000) and PAVAS (MW20,000) achieved a reduction of approximately 50% in the number of giantcells.

On the other hand, and in marked contrast thereto, dextran sulphate wasnot inhibitory to giant cell formation at a concentration of 4 μg/ml.This dextran sulphate required a concentration of 100 ug/ml tocompletely suppress giant cell formation.

In another series of experiments, PAVAS (MW 10,000) and PAVAS (MW20,000) and PVAS (MW 20,000) also inhibited the giant cell formationinduced by HIV-2, at concentrations similar to those required forinhibition of giant cell formation induced by HIV-1.

Thus, PAVAS and PVAS are potent inhibitors of giant cell formationinduced by HIV-1 and HIV-2.

EXAMPLE 5 Inhibition of Virus Adsorption

The inhibitory effect of the test compounds on virus adsorption wasmeasured by an indirect immunofluorescence/laser flow cytographic methodwhich had been established for this purpose.

In this test, MT-4 cells were exposed to HIV-1 virions which had beenconcentrated from the supernatant of HIV-1-infected HUT-78 cells, in thepresence or absence of test compounds. After 30 minutes incubation at37° C., the cells were thoroughly washed with phosphate-buffered saline(at 4° C.) to remove unadsorbed virus particles. Then, the cells wereprocessed for indirect immunofluorescence using polyclonal antibody toHIV-1 and analysed for cell-bound HIV-1 virions by laser flowcytofluorography. A control test was effected with MT-4 cells withoutexposing them to HIV-1 virions.

The results are represented in the fluorescence histograms of FIG. 3which have been obtained by plotting the relative cell number belongingto a certain intensity of green fluorescence against the logarithm ofthat intensity.

Solid-line histograms represent the fluorescence of MT-4 cells in thecontrol tests, while dotted-line histograms represent the fluorescenceof MT-4 cells in the actual tests. Panels A, B and C refer to testswithout any test compound, to tests with dextran sulphate (MW 5,000) at20 μg/ml, and to tests with PAVAS (MW 20,000) at 4 μg/ml, respectively.

It can be noted from panel A that, in the absence of any test compound,there is a clear difference in fluorescence properties between MT-4cells exposed to HIV-1 virions and MT-4 cells not exposed thereto. Infact, exposure to HIV-1 virions results in a shift of the histogram tohigher fluorescence intensities.

Panel B shows that, if the tests are carried out in the presence of 20μg/ml dextran sulphate, both histograms will coincide, meaning thatthere is no difference anymore between the fluorescence properties ofMT-4 cells exposed or not exposed to HIV-1 virions. This means that thebinding of HIV-1 virions to MT-4 cells is completely blocked by 20 μg/mlof dextran sulphate.

In the same way, panel C shows that virus adsorption onto the MT-4 cellscan be suppressed completely by 4 μg/ml of PAVAS (MW 20,000).

The same results could be achieved with PAVAS (MW 10,000) and with PVAS(MW 20,000) (data not shown).

What we claim is:
 1. A method for the treatment of immunodeficiencydiseases causally related to human immunodeficiency virus, whichcomprises administering an amount of a sulfated vinyl polymer selectedfrom the group consisting of sulfated polyvinylalcohols, sulfatedcopolymers of (meth)acrylic acid and vinylalcohol, together with atleast one pharmaceutically acceptable excipient, in an amount effectiveto treat a patient suffering from said immunodeficiency disease.
 2. Themethod as set forth in claim 1, wherein said sulfated vinyl polymer is asulfated copolymer of acrylic acid and vinylalcohol in a monomer ratioof about 1:9.
 3. The method as set forth in claim 2, wherein saidsulfated copolymer has a molecular weight of about 10,000 and asulfation degree of about 60%.
 4. The method as set forth in claim 2,wherein said sulfated copolymer has a molecular weight of about 20,000and a sulfation degree of about 50%.
 5. The method as set forth in claim1, wherein said sulfated vinyl polymer is a sulfated polyvinylalcoholhaving a molecular weight of about 20,000 and a sulfation degree ofabout 50%.
 6. The method as set forth in any of claims 1-5, wherein saidsulfated vinyl polymer is used in a concentration of 0.1 to 100%.
 7. Themethod as set forth in any of claims 1-5, wherein said sulfated vinylpolymers are administered in powder, suspension, solution, spray,emulsion, unguent or cream form.